Epidemic dengue caused by one or more of the four dengue virus serotypes continues to pose a major public health problem in most tropical and subtropical area. A safe and effective dengue vaccines against dengue are currently still not available. A series of DEN4 mutants containing a deletion in the 5 or 3 non-coding (NC) region were constructed. Growth analysis indicated that most of these DEN4 deletion mutants exhibited reduced replicative capacity in simian cell culture and in primates. Such DEN4 deletion mutants might prove to be useful for immunization of humans against dengue. The simian Vero cell line that was certified for production of human vaccines was selected for growth of DEN4 vaccine candidates to be studied in the clinical trials. DEN4 mutant 3?d 172-143 was passaged three times in Vero cells in serum-free medium to prepare a pre-vaccine pool from which the final vaccine lot was produced. Further concentration and purification of virus was not necessary as the level of cell DNA in the preparation was sufficiently low enough to satisfy FDA requirements. These clinical vaccine studies will be performed in collaboration with Drs. Brian Murphy and Robert Chanock (LID, NIAID), and Dr. Ruth Karon (Johns Hopkins University). The complete nucleotide sequence of the genome of the DEN4 candidate vaccine was also determined. There were only two conservative amino acid changes, compared to that present in the original DEN4 3?d 172-143 cDNA clone: Val to Ala at position 109 of NS4B and Lys to Arg at position 249 of NS5. These amino acid differences were probably adventitous mutations that had occurred during virus recovery and subsequent preparation of the candidate vaccine. Extensive safety tests have been completed for the bulk vaccine preparation as well as the final vaccine container materials. Following successful completion of these tests that are required for FDA approval, we have recently filed an IND for clinical trials in healthy adults to begin this fall. - Dengue type 4 virus cDNA, deletion mutants, candidate vaccines, Vero cells, infectivity and immunogencity in primates